S-adenosylhomocysteine (SAH) hydrolase is a ubiquitous cellular enzyme catalyzing the hydrolysis of SAH to adenosine (Ado) and homocysteine (Hcy). SAH hydrolase has been an attractive therapeutic target for a number of medical indications including antiviral, anticancer, anti-inflammation, immunosuppression, and plasma Hcy-lowering for prevention or treatment of cardiovascular diseases due to its central role in regulation of biological methylation reactions. Yuan et al., Exp. Opin. Ther. Patents, 9: 1197-1206 (1999); Yuan et al., in Adv. Antiviral Drug Des. vol 2, pp. 41-88, De Clercq (ed)., JAI Press, Inc. London, UK (1996). Inhibition of SAH hydrolase results in inhibition of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. For example, inhibition of SAH hydrolase results inhibition of viral mRNA methylation, thus inhibiting viral replication (Scheme 1).

Numerous inhibitors of SAH hydrolase have been identified from naturally occurring compounds and synthetic compounds, including irreversible and reversible inhibitors. See, e.g., Yuan et al., Exp. Opin. Ther. Patents, 9: 1197-1206 (1999); Wolfe and Borchardt, Journal of Medicinal Chemistry, 34:1521-1530 (1991); Votruba and Holy, Coll. Czech. Chem. Commun., 45:3039 (1980); Schanche et al., Molecular Plarmacology, 26:553-558 (1984); U.S. Ser. No. 10/410,879. It is an object of the invention to provide methods for screening inhibitors of SAH hydrolase.
S-adenosylmethionine (SAM)-dependent methyltransferase is an enzyme that catalyzes the transfer of a methyl group from SAM to a substrate and converts SAM to SAH. Methyltransferase, including SAM-dependent methyltransferase catalyzing abnormal methylation has been linked to pathological conditions (see, e.g., U.S. Pat. No. 5,876,996). For example, covalent modification of cellular substrates with methyl groups has been implicated in the pathology of cancer and other diseases (Gloria, et al., Cancer, 78:2300-2306 (1996)). Cytosine hypermethylation of eukaryotic DNA prevents transcriptional activation (Turker and Bestor, Mutat. Res., 386:119-130 (1997)). N6-methyladenosine is found at internal positions of mRNA in higher eukaryotes (Bokar, et al., J. Biol. Chem., 269:17697-17704 (1994)). Hypermethylated viral DNA is transcribed at higher rates than hypo- or hemimethylated DNA in infected cells (Willis, et al. Cell. Biophys., 15:97-111 (1989)).
In addition, many pathways of small molecule degradation, such as those of neurotransmitters, require methyltransferase activity (U.S. Pat. No. 5,876,996; and Kagan and Clarke, Arch. Biochem. Biophys., 310:417427 (1994)). Degradation of catecholamines (epinephrine ornorepinephrine) requires phenylethanolamine N-methyltransferase. Hydroxyindole methyltransferase converts N-acetyl-5-hydroxytryptamine to melatonin in the pineal gland.
In their roles as a rate-limiting step in methyltransferase reactions, SAM-dependent methyltransferases have been identified as targets for psychiatric, antiviral, anticancer and anti-inflammatory drug design (U.S. Pat. No. 5,876,996). For instances, sequence-specific methylation inhibits the activity of the Epstein-Barr virus LMP1 and BCR2 enhancer-promoter regions (Minarovits et al., Virology, 200:661-667 (1994)). 2′-5′-linked oligo(adenylic acid) nucleoside analogues synthesized by interferon-treated mouse L cells act as antiviral agents (Goswarmi, et al., J. Biol. Chem., 257:6867-6870 (1982)). Adenine analog inhibitors of AdoMet-MT decreased nucleic acid methylation and proliferation of leukemia L1210 cells (Kramer et al., Cancer Res., 50:3838-3842 (1990)). Therefore, another object of the invention is to provide methods for screening for inhibitors for SAM-dependent methyltransferases.